Rb and p130 control cell cycle gene silencing to maintain the postmitotic phenotype in cardiac myocytes

Both Rb and p130 are required for the recruitment of heterochromatin proteins that mediate silencing of proliferation genes in adult cardiac myocytes

Epigenetic regulation of myogenic gene expression by heterochromatin protein 1 alpha

Heterochromatin protein 1 (HP1) is an essential heterochromatin-associated protein typically involved in the epigenetic regulation of gene silencing. However, recent reports have demonstrated that HP1 can also activate gene expression in certain contexts including differentiation. To explore the role of each of the three mammalian HP1 family members (α, β and γ) in skeletal muscle, their expression was individually disrupted in differentiating skeletal myocytes. Among the three isoforms of HP1, HP1α was specifically required for myogenic gene expression in myoblasts only. Knockdown of HP1α led to a defect in transcription of skeletal muscle-specific genes including Lbx1, MyoD and myogenin. HP1α binds to the genomic region of myogenic genes and depletion of HP1α results in a paradoxical increase in histone H3 lysine 9 trimethylation (H3K9me3) at these sites. JHDM3A, a H3K9 demethylase also binds to myogenic gene’s genomic regions in myoblasts in a HP1α-dependent manner. JHDM3A interacts with HP1α and knockdown of JHDM3A in myoblasts recapitulates the decreased myogenic gene transcription seen with HP1α depletion. These results propose a novel mechanism for HP1α-dependent gene activation by interacting with the demethylase JHDM3A and that HP1α is required for maintenance of myogenic gene expression in myoblasts

H3K9me3 levels on myogenic genes increased in C2C12 myoblasts after depleting HP1α.

<p><i>A.</i> Schematic diagram of the genomic structure of the mouse Lbx1 gene and locations of primers used for subsequent ChIP experiments. <i>B.</i> Protein-DNA complexes from cross-linked chromatin extracted from C2C12 myoblasts cultured in GM were immunoprecipitated with HP1α or mouse IgG. Bound DNA was amplified using the indicated PCR primers. <i>C, D,</i> C2C12 myoblasts were transfected with indicated siRNA, 48 hours after transfection, cross-linked chromatin was extracted and immunoprecipitated with indicted antibodies. <i>Lbx1</i> exon 2 (C) or <i>Lbx1</i> genomic sequences including exon 1, intron and exon 2 (D) were amplified. E. C2C12 myoblasts were transfected with the indicated siRNA, 48 hours after transfection total cell lysates were subjected to Western blotting with the indicated antibodies. <i>F.</i> C2C12 myoblasts were transfected with indicated siRNA, 48 hours after transfection cross-linked chromatin was extracted and immunoprecipitated with anti-H3K9me3 antibody. Precipitated DNA was used for PCR with primers spanning the MEF2-binding site on the myogenin gene promoter.</p

HP1α recruits JHDM3A to <i>Lbx1</i> gene and knockdown of JHDM3A in myoblasts impairs Lbx1, MyoD and myogenin expression.

<p><i>A.</i> C2C12 myoblasts were transfected with NSsiRNA or HP1αsiRNA, 48 hours after transfection ChIP assay was performed with anti-JHDM3A antibody. <i>Lbx1</i> exon 2 and myogenin gene promoter were amplified. <i>B.</i> C2C12 myoblasts were transfected with the indicated siRNA, 48 hours after transfection total cell lysates were subjected to Western blotting with the specified antibodies. <i>C, D</i> Immunoprecipitation (IP) was performed using nuclear extracts prepared from NIH3T3 cells over-expressing Flag-JHDM3A and GFP-HP1α with either anti-Flag (C) or anti-GFP antibodies (D). Immunoprecipitated complexes were blotted (IB) with indicated antibodies. <i>E, F.</i> Endogenous HP1 and JHDM3A interaction was examined using C2C12 myoblasts nuclear extract. Nuclear extracts were immunoprecipitated with anti-HP1α (E) or anti-JHDM3A (F) antibody, and western blotted for HP1α and JHDM3A. <i>G</i>, <i>In vitro</i> translated ³⁵S-JHDM3A protein was incubated with GST or GST-HP1α immobilized on the glutathione beads. Bound proteins were separated on SDS-PAGE and visualized by autoradiography. <i>H.</i> C2C12 myoblasts cultured in GM were transfected with NSsiRNA or JHDM3AsiRNA. After 48 hours, total RNA was isolated and expression of the indicated genes determined by semiquantitative PCR. <i>I.</i> C2C12 myoblasts were transfected with NSsiRNA or JHDM3AsiRNA, 48 hours after transfection cross-linked chromatin was extracted and immunoprecipitated with anti-H3K9me3 antibody. <i>Lbx1</i> exon 2 genomic sequences were amplified. <i>J.</i> C2C12 myoblasts were transfected with the indicated siRNA, 48 hours after transfection total cell lysates were subjected to Western blotting with the specified antibodies.</p

HP1α is required for skeletal muscle differentiation.

<p>C2C12 cells were transfected with the indicated siRNA in GM and 24 hours after transfection, DM was added and cultured for 72 hours. <i>A.</i> Phase contrast images were obtained (A a–d). Confocal fluorescence microscopy was performed after immunostaining for skeletal sarcomeric myosin heavy chain (red) and DAPI (blue) (A e–h). Scale bar equals 50 µm (A a–d) and 6 µm (A e–h). <i>B,</i> Total protein lysates were subjected to Western blotting. <i>C.</i> C2C12 cells were transfected with NSsiRNA or HP1αsiRNA (#1: ID# 60593; #2: ID# 60497) and cultured in DM for 72 hours. Total protein lysates were subjected to Western blotting. <i>D.</i> Reintroduction of siRNA resistant version of HP1α rescued myogenic differentiation in HP1αsiRNA transfected cells. C2C12 myoblasts were co-transfected with the indicated siRNA and pLPC-EHGF or pLPC-EHGF-rHP1α plasmids. 24 hours after transfection, DM was added and cultured for another 72 hours. Cells were stained with anti-GFP (green) and anti-Troponin C (red), nuclei were counterstained with DAPI (blue). Scale bar equals 50 µm. <i>E</i>. The total number of GFP positive cells and the cells positive for both GFP and Troponin C were counted and percentage of Troponin C positive cells in GFP positive cells were calculated. *<i>P</i><0.01 for siHP1α/GFP versus siHP1α/GFP-rHP1α cells. Expression of GFP and GFP fusion HP1 were detected by Western blotting using anti-GFP antibody.</p

HP1α-deficient myocytes demonstrate a defect at the committed myoblast stage with reduced Lbx and MyoD expression.

<p><i>A, B, C.</i> C2C12 myoblasts were transfected with indicated siRNA. 24 hours after transfection, transfection medium were changed to GM or DM, cells were incubated for an additional 24 hours. Total RNA was isolated and semiquantitative PCR analysis of gene expression was performed (A, C). mRNA levels of Lbx1 and MyoD (B) in cells cultured in GM quantified by real-time PCR normalized to GAPDH. *<i>P</i><0.05 for Lbx1 levels siNS vs. siHP1α; **<i>P</i><0.05 for MyoD levels siNS vs. siHP1α. <i>D</i>. EDU assay were performed 24 hours after indicated siRNA transfection in C2C12 myoblasts. Percentage of EDU positive cells was calculated. *<i>P</i><0.05 for sins versus siHP1α. <i>E.</i> C2C12 cells were transfected with indicated siRNA. Total RNA was isolated 36 hours after transfection. Semiquantitative PCR analysis of gene expression was performed. <i>F.</i> C2C12 myoblasts were co-transfected with indicated siRNA and GFP-expressing plasmid. Total RNA was extracted from GFP-positive cells isolated by fluorescence-activated cell sorting (FACS). Semiquantitative PCR analysis of gene expression was performed.</p

HP1α is dispensable for maintenance of terminal differentiation.

<p>C2C12 cells were cultured in DM for 72 hours and then transfected with indicated siRNA. <i>A</i>, 48 hours after transfection, total RNA was isolated and semiquantitative PCR analysis of gene expression was performed. <i>B.</i> 24 hours after siRNA transfection, DMEM contain 20% FBS were added and cells were cultured for another 24 hours. Total protein were extracted and probed for indicated antibodies. <i>C.</i> 24 hours after siRNA transfection, cells were stimulated with DMEM contain 20% FBS in the presence of BrdU for 24 hours. Cell cycle reentry was determined by immunostaining for BrdU incorporation (Blue: DAPI, Red: sarcomeric myosin heavy chain; Green; BrdU). Scale bar equals 50 µm.</p

Expression and nuclear distribution of HP1 proteins during skeletal muscle differentiation.

<p><i>A.</i> C2C12 cells were cultured in GM (growth medium) or DM (differentiation medium) and collected at indicated time points. Total protein was extracted and subjected to Western blotting. <i>B</i>. Confocal fluorescence microscopy was performed on C2C12 myoblasts and myotubes after immunostaining for the indicated HP1 protein (green) and DAPI (converted to red). Scale bar equals 10 µm.</p